A phosphate and calcium-enriched diet promotes progression of 5/6-nephrectomy-induced chronic kidney disease in C57BL/6 mice

Abstract

C57BL/6 mice are known to be rather resistant to the induction of experimental chronic kidney disease (CKD) by 5/6-nephrectomy (5/6-Nx). Here, we sought to characterize the development of CKD and its cardiac and skeletal sequelae during the first three months after 5/6-Nx in C57BL/6 mice fed a calcium- and phosphate enriched diet (CPD) with a balanced calcium/phosphate ratio. 5/6-NX mice on CPD showed increased renal fibrosis and a more pronounced decrease in glomerular filtration rate when compared to 5/6-Nx mice on normal diet (ND). Interestingly, despite comparable levels of serum calcium, phosphate, and parathyroid hormone (PTH), circulating intact fibroblast growth factor-23 (FGF23) was 5 times higher in 5/6-Nx mice on CPD, relative to 5/6-Nx mice on ND. A time course experiment revealed that 5/6-Nx mice on CPD developed progressive renal functional decline, renal fibrosis, cortical bone loss, impaired bone mineralization as well as hypertension, but not left ventricular hypertrophy. Collectively, our data show that the resistance of C57BL/6 mice to 5/6-Nx can be partially overcome by feeding the CPD, and that the CPD induces a profound, PTH-independent increase in FGF23 in 5/6-Nx mice, making it an interesting tool to assess the pathophysiological significance of FGF23 in CKD.

Figure 1

Calcium and phosphate-enriched diet promotes renal functional decline in 5/6-nephrectomized C57BL/6 mice. (a) Body weight, (b) serum calcium, (c) serum phosphate, (d) serum parathyroid hormone (PTH), (e) serum intact fibroblast growth factor-23 (FGF23), (f) serum creatinine, (g) serum urea, (h) glomerular filtration rate per g body weight (GFR/BW) expressed as percent of Sham control on same diet, (i) percent picrosirius red-stained area in kidney, (j) representative images of renal picrosirius red staining to assess renal fibrosis, (k) systolic and (l) mean arterial pressure measured by intraarterial catheterization, and (m) ejection fraction measured by echocardiography in sham-operated (Sham) and 5/6-Nx (NX) C57BL/6 mice on CPD and ND, 12 weeks postsurgery. N = 4–12 per group. Bars depict mean values ± SD. Data were analyzed using 2-way ANOVA, followed by Student’s t test. Insets show results of 2-way ANOVA. *P < 0.05; ***P < 0.001 versus Sham on same diet by Student’s t test. In (a) results of Student’s t test are shown only for the 4-, 8-, and 12-week time points (boxes, P < 0.05 vs. Sham on ND; circles, P < 0.001 vs. Sham on CPD). Bar = 100 µm in (j).

Figure 2

Progressive renal functional decline in 5/6-Nx mice on CPD. (a) Serum creatinine, (b) serum urea, (c) glomerular filtration rate per g body weight (GFR/BW) expressed as percent of Sham control at same time point, (d) serum calcium, (e) serum parathyroid hormone (PTH), (f) serum aldosterone, (g) serum intact fibroblast growth factor-23 (FGF23), (h) percent picrosirius red-stained area in kidney, (i) mean glomerulosclerosis score, (j) representative images of renal picrosirius red staining to assess renal fibrosis, and (k) representative images of renal PAS staining to assess glomerulosclerosis in Sham-operated (SH) and 5/6-Nx (NX) C57BL/6 mice on CPD, 4, 8 and 12 weeks postsurgery. N = 4–12 mice per group. Bars depict mean values ± SD. Data were analyzed using 2-way ANOVA, followed by Student’s t-test. Insets show results of 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001 versus Sham at same time point by Student’s t test. Bar = 100 µm in (j) and 50 µm in (k), respectively.

Figure 3

Mild hypertension but absence of left ventricular hypertrophy and dysfunction in 5/6-Nx mice on CPD. (a) Systolic, (b) mean arterial, and (c) pulse pressure measured by intraarterial catheterization, (d) ejection fraction and (e) left ventricular anterior wall thickness measured by echocardiography, and (f) cardiomyocyte size measured by (g) wheat germ agglutinin staining in Sham-operated (SH) and 5/6-Nx (NX) C57BL/6 mice on CPD, 4, 8 and 12 weeks postsurgery. N = 3–11 mice per group. Bars depict mean values ± SD. Data were analyzed using 2-way ANOVA, followed by Student’s t test. Insets show results of 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001 versus Sham at same time point by Student’s t test. Bar = 50 µm in (g).

Figure 4

Reduced bone mass and impaired bone mineralization in 5/6-NX mice on CPD. (a) Proximal tibial total bone mineral density (BMD), (b) tibial diaphyseal total BMD, and (c) vertebral total BMD measured by pQCT; (d) osteoid volume, (e) osteoid surface, (f) osteoclast surface, and (g) mineralization lag time measured in tibial cancellous bone by histomorphometry; (h) serum alkaline phosphatase activity, and (i) urinary deoxypyridinoline/creatinine (UrDPD/Crea) excretion in Sham-operated (SH) and 5/6-Nx (NX) C57BL/6 mice on CPD, 4, 8 and 12 weeks postsurgery. N = 4–12 mice per group. Bars depict mean values ± SD. Data were analyzed using 2-way ANOVA, followed by Student’s t test. Insets show results of 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001 versus Sham at same time point by Student’s t test.